Isolation and characterisation of an aryl-β-D-glucoside uptake and utilisation system ( abg ) from the gram-positive ruminal Clostridium species C. longisporum

A phosphotransferase-dependent aryl-β-glucoside uptake and utilisation system (abg) was isolated from the ruminal Clostridium (“C. longisporum”). The system is composed of three genes, abgG, abgF and abgA, and a number of regulatory regions, including terminator/antiterminator type stem-loop structures preceding the abgG and abgF genes. Similarity analysis of the proteins encoded by these genes indicated that they were responsible for the regulation of the abg system through antitermination (AbgG), the uptake and phosphorylation of aryl-β-glucosides (AbgF) and the hydrolysis of the intracellular phosphorylated glycosides (AbgA). Experimental evidence for the functions of AbgF and AbgA was obtained. Although it was not possible to demonstrate any function for AbgG, a promoter 5′ to the abgG gene was identified which was responsible for expression of the downstream genes. The abg system is remarkably similar to operons from the gram negative Enterobacteriaceae, both in the coding and non-coding regulatory regions. Received: 3 April 1997 / Accepted: 8 September 1997     

Subtle re-location of dendritic spine branches containing membrane with fast spiking mechanisms can alter synaptic efficacy

The potential physiological impact of morphological changes in the active dendritic spines, which are believed to be associated with altered synaptic efficacy, was investigated in a computer simulation study using the NEURON package [1]. A compartmental model of a simplified neuron was built, which included 30 complex spines (neck, head, and active zone) and accommodating AMPA-type synaptic inputs with alpha-function conductances. Hodgkin-Huxley type excitable membranes were inserted into the spine heads. It was shown that arranging spines in dense clusters, as opposed to a uniformly random spine distribution, has a negligible effect on the synaptic signal transfer (other model conditions, including synaptic input and spine density, remained unchanged). However, if a proportion (e.g., 3–20%) of the spines partly fuse with their neighbors forming branched spines, this could increase dramatically the cell response to the unchanged synaptic input. Results of this pilot study provide the basis for a more detailed investigation of the relationship between the spine arrangement and synaptic function, considering dual-component synaptic currents and mechanisms controlling ion fluxes in the dendritic compartments.     

Steady-state kinetics of F1-ATPase. Mechanism of anion activation

The kinetic behaviour of the ATPase activity of beef heart F1 depends largely on the exposure of the enzyme to some anionic ligands such as sulphate and/or EDTA. F1 prepared in the presence of such anions exhibited a triphasic kinetic pattern whereas F1 from which those anions were removed by dialysis exhibited only two Km values for ATP. Conversely to what has been previously reported, bicarbonate did not linearize F1-ATPase kinetics. Moreover, anion activation cannot be simply explained by promotion of ADP release but mainly by an increase in affinity of the third catalytic site for ATP.     

Two-dimensional crystallization of bovine rhodopsin

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2(1) with dimensions of 8.3 X 7.9 nm and interaxis angles of 86 +/- 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20-40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corless, J.M., McCaslin, D.R. and Scott, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116-1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz, E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128-131). Crystallization was accomplished by lowering the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH4)2SO4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.     

A comparison of the receptor constants of morphine and ethylketocyclazocine for analgesia and inhibition of gastrointestinal transit in the rat

The efficacies and dissociation constants of proposed mu and kappa receptor agonists (morphine and ethylketocyclazocine, respectively) were compared using the method of partial irreversible blockade (with buprenorphine) and Stephenson's theory of drug action. While there was good agreement between the dissociation constant (KA) of morphine in analgesia (3.3 x 10(-5) M) and in inhibition of gastrointestinal transit (1.1 x 10(-5) M), the KA of ethylketocyclazocine differed by an order of magnitude in these endpoints (3.2 x 10(-6) M and 6.7 x 10(-5) M, respectively). The efficacies of morphine were found to be similar for the two effects studied (4.23 and 5.26), while those for ethylketocyclazocine differed markedly (2.06 and 10.39). The fraction of receptors remaining unblocked after buprenorphine was consistent for the test but not for the agonist, indicating a different distribution of receptors for the two endpoints. Our results strongly suggest that morphine induces analgesia, and slows transit in the small intestine, through the same type of receptor. The same conclusion cannot be drawn for ethylketocyclazocine.     

Immunohistochemical evaluation of ras oncogene expression in pulmonary and pleural neoplasms

We undertook an immunohistochemical analysis of human bronchopulmonary epithelial neoplasms and pleural mesotheliomas using a monoclonal antibody which recognizes ras oncogene products (p21ras). The monoclonal antibody, RAP-5, recognizes both unaltered and certain mutated p21ras. Formalin fixed and paraffin embedded tissue samples of 187 lung epithelial tumors and 27 pleural mesotheliomas were investigated; normal and bronchiectatic lungs were similarly studied. Normal lung and pleural tissue did not immunostain except for occasional type II pneumocytes. Reactive type II pneumocytes adjacent to carcinomas and bronchiectasis immunostained consistently. Twenty four/34 (71%) squamous carcinomas immunostained. Only 8/50 (16%) adenocarcinomas immunostained focally and weakly whereas 19/24 (79%) bronchioloalveolar carcinomas immunostained. Eleven/18 (61%) large cell carcinomas immunostained with variable intensity. Eleven/13 (85%) carcinoids, 6/7 (85%) well differentiated neuroendocrine carcinomas, and 18/21 (86%) intermediate cell neuroendocrine carcinomas immunostained while none of 20 small cell neuroendocrine carcinomas immunostained. Only a few mesotheliomas were immunostained focally. Two/14 (14%) epithelial type and 1/9 (11%) biphasic type mesotheliomas immunostained weakly; none of 4 spindle cell mesotheliomas immunostained. We conclude that while at least occasional cases of most types of pulmonary epithelial neoplasms express p21ras, the frequency and intensity of the expression are distinctly greater in certain tumor types such as squamous, bronchioloalveolar, and neuroendocrine neoplasm except for the small cell type. Contrary to these lung epithelial neoplasms, most mesotheliomas did not immunostain for p21ras. Whether the enhanced p21ras expression may point to a different mechanism of transformation or may merely reflect differentiation features remains undetermined.     

Interaction of the organic anion 1-aniline 8-naphthalene sulfonate (ANS) with isolated rat hepatocytes

The interaction of ANS with rat hepatocytes in time was studied by fluorescence spectroscopy. The intercept of the first linear portion of the time curve of interaction showed a positive value over all the ANS concentration range employed. This value was maintained after cellular disruption by homogenization. It was affected by ionic strength, pH, and divalent cation in the incubation medium, all conditions affecting the cellular surface. These data suggest that this phenomenon might be a binding of the compound to the hepatocytes surface. Due to the time constant and its disappearance after cellular disruption the other slower component of the curve seems to correspond to a process of translocation across the membrane.     

Deoxyuridine triphosphatase: A potential site of interaction with pyrimidine nucleotide analogues

Deoxyuridine triphosphatase has been purified from cultured human lymphoid cells in high yield and stable form by a relatively simple procedure. The properties differed somewhat from those reported previously, e.g. apparent K_m, molecular weight, and effects of divalent metals. No other naturally occurring dNTP or NTP serves as substrate, however, the enzyme may be an important site of interaction with intracellular derivatives of analogues of dUrd. It is shown here that deoxyuridine triphosphatase acts on araUTP, 6-azadUTP, 2′-FdUTP, and 2′,3′-dideoxyUTP, but the enzyme has no effect on 5-C1dUTP, 5-BrdUTP, 5-HgdUTP and dUrd-5′[α-thio]triphosphate. For the preparation of one of the analogues, the enzyme, trans-N-deoxyribosylase, from Lactobacillus, was used to prepare the deoxynucleoside from the base, a procedure that may have general usefulness.